Investigation of the carboligase activity of thiamine diphosphate-dependent enzymes using kinetic modeling and NMR spectroscopy

Kokova, Mariya; Zavrel, Michael; Tittmann, Kai; Spieß, Antje C.; Pohl, Martina

doi:10.1016/j.molcatb.2009.02.021

Cited by 23 publications

(22 citation statements)

References 18 publications

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“…Especially the formation and cleavage of CC bonds is important as it leads to the formation of enantiopure 2‐hydroxy ketones, which can easily be converted into valuable products like amino alcohols or amines . Benzaldehyde lyase from Pseudomonas fluorescens ( Pf BAL) is a ThDP‐dependent enzyme with high activity and excellent enantioselectivity, which catalyzes the formation and cleavage of the acyloin linkage of two molecules of benzaldehyde, resulting in ( R )‐benzoin . Furthermore, both aromatic and aliphatic aldehydes can be used as substrates for formation of asymmetric hydroxy ketones …”

Section: Introductionmentioning

confidence: 99%

Simultaneous identification of reaction and inactivation kinetics of an enzyme‐catalyzed carboligation

Ohs

Leipnitz

Schöpping

et al. 2018

Biotechnology Progress

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Thiamine diphosphate (ThDP)-dependent enzymes catalyze a broad range of reactions with excellent enantioselectivity. Among these reactions, carboligations of aldehydes are of particular interest since the products, chiral hydroxy ketones, are valuable building blocks in the pharmaceutical industry. However, the substrates, for example, benzaldehyde, inactivate the biocatalysts, for example the ThDP-dependent benzaldehyde lyase from Pseudomonas fluorescens (PfBAL). Because only few mechanistic kinetic models for carboligation and simultaneous inactivation are available today, we quantitatively determined the reaction kinetics and inactivation of the self-carboligation of benzaldehyde yielding the product (R)-benzoin catalyzed by PfBAL directly from progress curves using model-based experimental analysis. Discrimination of several inactivation models identified the substrate-dependent inactivation by benzaldehyde to be significant. Sensitivity analysis and optimal experimental design improved parameter precision significantly, to between 4 and 26% relative standard deviation while maintaining the necessary number of 13 experiments moderate. The developed mechanistic kinetic model will enable to perform a model-based process optimization to circumvent the substrate-dependent enzyme inactivation. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1081-1092, 2018.

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Section: Introductionmentioning

confidence: 99%

Simultaneous identification of reaction and inactivation kinetics of an enzyme‐catalyzed carboligation

Ohs

Leipnitz

Schöpping

et al. 2018

Biotechnology Progress

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“…As already mentioned above, especially the behaviours of benzaldehyde lyase from Pseudomonas fluorescens ( Pf BAL5,24) and benzoylformate decarboxylase from Pseudomonas putida ( Pp BFD) in monophasic buffer-organic solvent mixtures have been examined 24a. However, these studies only focused on the syntheses of benzoin and HPP, respectively.…”

Section: Introductionmentioning

confidence: 99%

Influence of Organic Solvents on Enzymatic Asymmetric Carboligations

et al. 2012

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The asymmetric mixed carboligation of aldehydes with thiamine diphosphate (ThDP)-dependent enzymes is an excellent example where activity as well as changes in chemo- and stereoselectivity can be followed sensitively. To elucidate the influence of organic additives in enzymatic carboligation reactions of mixed 2-hydroxy ketones, we present a comparative study of six ThDP-dependent enzymes in 13 water-miscible organic solvents under equivalent reaction conditions. The influence of the additives on the stereoselectivity is most pronounced and follows a general trend. If the enzyme stereoselectivity in aqueous buffer is already >99.9% ee, none of the solvents reduces this high selectivity. In contrast, both stereoselectivity and chemoselectivity are strongly influenced if the enzyme is rather unselective in aqueous buffer. For the S-selective enzyme with the largest active site, we were able to prove a general correlation of the solvent-excluded volume of the additives with the effect on selectivity changes: the smaller the organic solvent molecule, the higher the impact of this additive. Further, a correlation to log P of the additives on selectivity was detected if two additives have almost the same solvent-excluded volume. The observed results are discussed in terms of structural, biochemical and energetic effects. This work demonstrates the potential of medium engineering as a powerful additional tool for varying enzyme selectivity and thus engineering the product range of biotransformations. It further demonstrates that the use of cosolvents should be carefully planned, as the solvents may compete with the substrate(s) for binding sites in the enzyme active site.

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“…Therefore, it has to be assumed that MTBE hinders the intrinsic enzyme catalytic steps and compromises the performance of the enzyme, although the conclusion on distinct solvent effects is difficult in multi‐component reaction systems. Considering the stabilizing effect of MTBE on Lb ADH, however, our data suggest that there might be an enzyme–solvent interaction that bind within the active center or impair the enzyme flexibility that is important for the catalytic activity of alcohol dehydrogenases . Further data obtained under addition of both other substrates and solvents will be required to verify this assumption and to further allow for distinction of individual solvent effects.…”

Section: Resultsmentioning

confidence: 83%

Thermodynamic activity‐based intrinsic enzyme kinetic sheds light on enzyme–solvent interactions

Grosch

Wagner

Nistelkas

et al. 2016

Biotechnology Progress

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The reaction medium has major impact on biocatalytic reaction systems and on their economic significance. To allow for tailored medium engineering, thermodynamic phenomena, intrinsic enzyme kinetics, and enzyme-solvent interactions have to be discriminated. To this end, enzyme reaction kinetic modeling was coupled with thermodynamic calculations based on investigations of the alcohol dehydrogenase from Lactobacillus brevis (LbADH) in monophasic water/methyl tert-butyl ether (MTBE) mixtures as a model solvent. Substrate concentrations and substrate thermodynamic activities were varied separately to identify the individual thermodynamic and kinetic effects on the enzyme activity. Microkinetic parameters based on concentration and thermodynamic activity were derived to successfully identify a positive effect of MTBE on the availability of the substrate to the enzyme, but a negative effect on the enzyme performance. In conclusion, thermodynamic activity-based kinetic modeling might be a suitable tool to initially curtail the type of enzyme-solvent interactions and thus, a powerful first step to potentially understand the phenomena that occur in nonconventional media in more detail. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:96-103, 2017.

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Investigation of the carboligase activity of thiamine diphosphate-dependent enzymes using kinetic modeling and NMR spectroscopy

Cited by 23 publications

References 18 publications

Simultaneous identification of reaction and inactivation kinetics of an enzyme‐catalyzed carboligation

Simultaneous identification of reaction and inactivation kinetics of an enzyme‐catalyzed carboligation

Influence of Organic Solvents on Enzymatic Asymmetric Carboligations

Thermodynamic activity‐based intrinsic enzyme kinetic sheds light on enzyme–solvent interactions

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